RESUMO
Leukemia inhibitory factor (LIF) has been recognized as a novel inflammatory modulator in inflammation-associated diseases. This study aimed to investigate the modulation of LIF in dental pulp inflammation. Experimental pulpitis was established in wild-type (WT) and Lif-deficient (Lif-/-) mice. Histological and immunostaining analyses were conducted to assess the role of LIF in the progression of pulpitis. Mouse macrophage cell line (RAW264.7) was treated with LPS to simulate an inflammatory environment. Exogenous LIF was added to this system to examine its modulation in macrophage inflammatory response in vitro. Primary bone marrow-derived macrophages (BMDMs) from WT and Lif-/- mice were isolated and stimulated with LPS to confirm the effect of Lif deletion on macrophage inflammatory response. Supernatants from LIF and LPS-treated human dental pulp cells (hDPCs) were collected and added to macrophages. Macrophage chemotaxis was assessed using transwell assays. The results showed an increased expression of LIF and LIFR with the progression of pulpitis, and LIFR was highly expressed in macrophages. Lif deficiency alleviated experimental pulpitis with the reduction of pro-inflammatory cytokines and macrophage infiltration. Exogenous LIF promoted inflammatory response of LPS-induced macrophages through a STAT3/p65-dependent pathway. Consistently, Lif deletion inhibited macrophage inflammatory response in vitro. Supernatants of LIF-treated hDPCs enhanced macrophage migration in LPS-induced inflammatory environment. Our findings demonstrated that LIF aggravates pulpitis by promoting macrophage inflammatory response through a STAT3/p65-dependent pathway. Furthermore, LIF plays a crucial role in driving the recruitment of macrophages to inflamed pulp tissue by promoting chemokine secretion in DPCs.
Assuntos
Pulpite , Animais , Humanos , Camundongos , Polpa Dentária/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Fator Inibidor de Leucemia/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Pulpite/metabolismoRESUMO
OBJECTIVE: Fusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. METHODS: In this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 µg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. RESULTS: Proteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1ß, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1ß, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). CONCLUSIONS: Taken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.
Assuntos
Proteínas de Membrana , NF-kappa B , Camundongos , Animais , Bovinos , NF-kappa B/metabolismo , Proteínas de Membrana/metabolismo , Fusobacterium necrophorum/genética , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6 , Fator 88 de Diferenciação Mieloide/metabolismo , Proteômica , Citocinas/metabolismo , RNA MensageiroRESUMO
Colovesical fistula (CVF) is usually developed from colonic diverticulitis, followed by tumor. Traditional surgery is usually completed in one or more stages. For complex cancerous CVF, radical resection is more difficult. We report a 62-year-old male patient diagnosed with sigmoid colon cancer combined with sigmoid vesical fistula. In the course of treatment, in addition to conventional surgery, neoadjuvant chemotherapy (NAC) was innovatively used. The sigmoid tumor and fistula were significantly shrunken. Radical surgery achieved negative margins.
RESUMO
Coronavirus subverts the host cell cycle to create a favorable cellular environment that enhances viral replication in host cells. Previous studies have revealed that nucleocapsid (N) protein of the coronavirus porcine epidemic diarrhea virus (PEDV) interacts with p53 to induce cell cycle arrest in S-phase and promotes viral replication. However, the mechanism by which viral replication is increased in the PEDV N protein-induced S-phase arrested cells remains unknown. In the current study, the protein expression profiles of PEDV N protein-induced S-phase arrested Vero E6 cells and thymidine-induced S-phase arrested Vero E6 cells were characterized by tandem mass tag-labeled quantitative proteomic technology. The effect of differentially expressed proteins (DEPs) on PEDV replication was investigated. The results indicated that a total of 5709 proteins, including 20,560 peptides, were identified, of which 58 and 26 DEPs were identified in the PEDV N group and thymidine group, respectively (P < 0.05; ratio ≥ 1.2 or ≤ 0.8). The unique DEPs identified in the PEDV N group were mainly involved in DNA replication, transcription, and protein synthesis, of which 60S ribosomal protein L18 (RPL18) exhibited significantly up-regulated expression in the PEDV N protein-induced S-phase arrested Vero E6/IPEC-J2 cells and PEDV-infected IPEC-J2 cells (P < 0.05). Further studies revealed that the RPL18 protein could significantly enhance PEDV replication (P < 0.05). Our findings reveal a mechanism regarding increased viral replication when the PEDV N protein-induced host cells are in S-phase arrest. These data also provide evidence that PEDV maintains its own replication by utilizing protein synthesis-associated ribosomal proteins.
Assuntos
Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Chlorocebus aethiops , Vírus da Diarreia Epidêmica Suína/genética , Proteômica/métodos , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Suínos , Timidina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células Vero , Replicação ViralRESUMO
Fusobacterium necrophorum, a Gram-negative anaerobe, is an important bovine pathogen that causes hepatic abscesses, foot rot, mastitis and endometritis. We have previously shown that the 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in bacterial infections; however, the molecular mechanisms by which this protein mediates adhesion remain unclear. In this study, we investigated the role of 43 K OMP in F. necrophorum adhesion to bovine epithelial cells using 43 K OMP-deficient mutants, and identified the protein that interacts with 43 K OMP by immunoprecipitation-mass spectrometry. Our results indicated that the native 43 K OMP and recombinant 43 K OMP could bind to the cell membrane of MAC-T or bovine endometrial epithelial cells (BEECs). When F. necrophorum was preincubated with antibodies against the recombinant 43 K OMP or bovine epithelial cells were preincubated with 43 K OMP, the adhesion of F. necrophorum to MAC-T or BEECs decreased significantly (P<0.01). We successfully constructed a 43 K OMP-deficient strain (A25Δ43 K OMP) and bacterial attachment to MAC-T or BEECs was significantly higher with the F. necrophorum A25 strain than with mutant strain A25Δ43 K OMP (P<0.01). The deficiency of 43 K OMP reduced the binding of F. necrophorum to bovine epithelial cells by 90.5 %-94.9 %. Among the 39 potential differential proteins, fibronectin, collagen and myosin were selected as the target proteins, and direct interaction between 43 K OMP of F. necrophorum and fibronectin was demonstrated. Taken together, these results suggest that 43 K OMP plays a key role in adhesion of F. necrophorum to bovine epithelial cells through its interaction with fibronectin. These findings provide a theoretical basis for the pathogenic mechanism of F. necrophorum.
Assuntos
Doenças dos Bovinos , Pododermatite Necrótica dos Ovinos , Infecções por Fusobacterium , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Células Epiteliais , Feminino , Fibronectinas/metabolismo , Pododermatite Necrótica dos Ovinos/microbiologia , Infecções por Fusobacterium/microbiologia , Infecções por Fusobacterium/veterinária , Fusobacterium necrophorum/genéticaRESUMO
Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.
Assuntos
Antivirais/farmacologia , Proteínas do Nucleocapsídeo de Coronavírus/química , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Quercetina/análogos & derivados , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Animais , Antivirais/química , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/genética , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus/antagonistas & inibidores , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/genética , Simulação de Acoplamento Molecular , Sinais de Localização Nuclear , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Quercetina/química , Quercetina/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/genética , Transdução de Sinais , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/genética , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The entry mechanism of porcine epidemic diarrhea virus (PEDV) remains unclear, especially the virus receptor. Our previous study revealed a potential correlation between integrin αvß3 and PEDV infection. In the current study, the effect of overexpression, silencing, antibody inhibition, and co-expression with porcine aminopeptidase N (pAPN) of integrin αvß3 on PEDV infection was investigated and analyzed in African green monkey Vero E6 cells and porcine intestinal epithelial cells (IECs) using the classical strain CV777 and variant strain HM2017 of PEDV. Integrin αvß3 significantly enhanced the replication of the classical and variant strains of PEDV in Vero E6 cells and IECs. The integrin αv and ß3 subunits were both involved in the enhancement of PEDV infection, the Arg-Gly-Asp peptides targeting integrin αvß3 significantly inhibited replication of PEDV in Vero E6 cells, and co-expression of integrin αvß3 with pAPN significantly enhanced replication of PEDV in Vero E6 and BHK-21 cells. These results demonstrate that integrin αvß3 enhances PEDV replication in Vero E6 cells and IECs. These data provide novel insights into the entry mechanism of PEDV.
Assuntos
Células Epiteliais/virologia , Integrina alfaVbeta3/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Cultura de Vírus/veterinária , Replicação Viral/fisiologia , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica , Inativação Gênica , Mucosa Intestinal/citologia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Células VeroRESUMO
Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P<0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the PEDV-infected Vero E6 cells, involving in integrin ß2/ß3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin ß2/ß3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions.
Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/veterinária , Proteínas Virais/metabolismo , Animais , Antígenos CD18/biossíntese , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Cistatina C/biossíntese , Expressão Gênica , Perfilação da Expressão Gênica , Integrina beta3/biossíntese , Vírus da Diarreia Epidêmica Suína/metabolismo , Proteômica/métodos , Suínos/virologia , Doenças dos Suínos/virologia , Células Vero , Proteínas Virais/biossínteseRESUMO
The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 6E6, which secretes the monoclonal antibody against PEDV S protein. The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/6E6). After sequence analysis, the scFv/6E6 gene was cloned into the prokaryotic expression vector pGEX-6p-1 with a GST-tag. The recombinant scFv/6E6 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the recombinant scFv/6E6 protein was purified from the inclusion body form by the gel-cutting measure followed by electroelution and dialysis. The recombinant scFv/6E6 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.